Table 6.

Troubleshooting guide for poor transfection efficiency

Problem encountered	Suggestion
Cell incompatibility	Check if PEI has been previously tested with your cell line. If not, change cell line if possible. We have successfully transfected GIV constructs with PEI in HEK293, HeLa, COS-7, NIH3T3, DLD1 and MDCK cell lines using the Basic Protocol 4.
DNA:PEI Ratio	We suggest using 1-2 μl of PEI per μg of DNA transfected. May need to be optimize for your cells.
Poor PEI quality	PEI could be kept at 4°C for 1 month maximum. However, if a decline of transfection efficiency is observed, we recommend taking out a new aliquot from −80°C.
High cell death	- Prolonged incubation with PEI could be toxic for the cells. We recommend an incubation of mix DNA/PEI for a maximum of 5h. - May need to adjust DNA/PEI ratio or decrease amount of DNA transfected.